Molecularly imprinted polymers for the treatment and diagnosis of medical conditions

ABSTRACT

Improved molecularly imprinted polymers (MIPs) with both higher and more specific binding capacity for particular bile acids and/or salts, including the synthesis of such MIPs, the compounds themselves, and specific applications thereof. As an example of a particularly preferred specific application of these compounds, the present invention encompasses the use of the MIPs as sequestrants in the gastrointestinal tract, particularly in order to bind and therefore remove toxins from the gastrointestinal tract. In addition, the present invention is also useful for treatment of various diseases which are related to, and/or characterized by, an effect of bile acids and salts, such as atherosclerosis, liver disease and various diseases of the gastrointestinal tract. The MIP compounds of the present invention are also useful for combination therapy with other medications and for diagnosis and monitoring of diseases.

[0001] This Application is a divisional application of U.S. patentapplication Ser. No. 09/893,643, filed on Jun. 29, 2001, which claimspriority from U.S. Provisional Application No. 60/215,882, filed on Jun.30, 2000, now expired, all of which are hereby incorporated as if fullyset forth herein.

FIELD OF THE INVENTION

[0002] The present invention is related to novel molecularly imprintedpolymers, and in particular to improvements in the production ofmolecularly imprinted polymers (MIPs), as well as to these specificMIPs, and to the use of MIPs for specific applications. The MIPs of thepresent invention are particularly suitable for binding to, and therebyremoving, toxins in the gastrointestinal tract. As an exemplaryimplementation, the present invention is described with regard to theremoval of bile acids and bile salts from the gastrointestinal tract. Inaddition, the present invention is also useful for treatment of variousdiseases which are related to, and/or characterized by an effect of,bile acids and bile salts, such as atherosclerosis, cancer, liverdisease and various diseases of the gastrointestinal tract. The MIPcompounds of the present invention are also useful for combinationtherapy with other medications. These medications may involve mechanismsof action that lower or change the composition of bile acids and saltsin the body or by a different mechanism.

[0003] In addition the present invention also is useful for thediagnosis and monitoring of various diseases by selectively binding toan established marker which is then identified using known bindingindicator techniques such as fluorescence. As an illustrative examplefor implementation, the present invention is described with regard tothe diagnosis of medical conditions which are related to, and orcharacterized by an effect of, bile acids and /or bile salts, such asatherosclerosis, various diseases of the gastrointestinal tract, cancerand inflammatory conditions. This is achieved by determining the levelof at least one specific bile acid or salt or the ratio of at least onespecific bile acid or to at least a second specific bile acid or saltand determining whether these levels fall within an establish rangewhich indicates the potential existence of the relevant disease. Theanalysis is performed on bile acids and or bile salts found in serum,bile, gastric contents, and feces.

BACKGROUND OF THE INVENTION

[0004] The subject of molecularly imprinted polymers has beenextensively reviewed (e.g., G. Wulff, Angew. Chem., Int. Ed. Engl. 1995,34, 1812-1832; A. G. Mayyes and K. Mosbach, Trends Anal. Chem. 1997, 16,321-332; E. N. Vulfson, C. Alexander, and M. J. Whitcombe Chem. Brit.1997, 33, 23-26; K. Haupt and K. Mosbach, Trends Biotechnol. 1998, 16,468-475; Molecular and Ionic Recognition with Imprinted Polymers, ACSSymp. Ser. 703; R. A. Bartsch and M. Maeda, Eds.; American ChemicalSociety, Washington, DC, 1998) and a number of patents on this topichave been issued [e.g., U.S. Pat. No. 4,127,730 (Wulff, G., Sarhan A.);U.S. Pat. No. 5,110,833 (Mosbach. K.); U.S. Pat. No. 5,630,978 (Domb,A.,); U.S. Pat. No. 5,587,273 (Yan, M. et al.); U.S. Pat. No. 5,872,198(Mosbach, K. et al;)].

[0005] A schematic depiction of the formation of MIPs for deoxycholicacid (DCA) and glycodeoxycholic acid (GDCA) is shown in FIGS. 1A and 1B.Although the binding/recognition site is actually a family ofnon-homogeneous sites, the scheme illustrates how the cavities for twosimilar substances may differ.

[0006] The synthesis of MIPs, including those described with regard tothe present invention in the “Description of the Preferred Embodiments”below, is performed with functional monomers. The monomers which wereused for the present invention include all of the monomers listed in thefollowing section. The synthesis of these monomers and relatedderivatives and analogs can be performed by organic chemists of ordinaryskill in the art. It should be noted that although the present inventionis described with regard to MIPs which bind bile acids and bile salts,this is for the purposes of description only and is not intended to belimiting in any way.

[0007] Bile acid sequestrants. A number of polymers, such ascholestyramine, are used as bile acid sequestrants. Their action isbased on the presence of strongly basic groups in the polymer(typically, ion exchange resin type of polymers) and they are used forcholesterol lowering and bile-related diseases. These materials arelimited because they have limited potency and they also remove (bind)other required substances such as nutrients, drugs, etc. In addition,they often irritate the bowel and are not convenient or palatable to thepatient. Accordingly there is a need for improved bile sequestrants.Selective MIPs which bind bile acids and salts do not remove needednutrients, drugs or other substances and will be more potent; they willhave low or no bowel irritation and have improved dosages. Importantly,the MIPs can be made so that they are selective to the more hydrophobicbile acids such as deoxycholic acid. Research shows that the morehydrophobic bile acids inhibit the removal of LDL from the blood streamand lead to a higher cholesterol blood serum level (Hueman, D. M. etal., J Lipid Res. 30: 1161, 1989).

[0008] While bile acids and salts serve important functions in the body,such as promoting digestion of fat, researchers have found that the morehydrophobic (water-resistant) bile acids, such as deoxycholic acid(DCA), chenodeoxycholic acid (CDCA) and lithocholic acid (LCA)facilitate higher absorption of lipids such as cholesterol and fats intothe blood stream and are toxic, causing damage to cells and promotingcancer. Current research indicates that these more hydrophobic bileacids are highly significant disease-causing agents.

[0009] Additionally, research has implicated specific bile acids andbile salts as contributing to a number of diseases. For example, DCA hasbeen implicated in the following:

[0010] Gallstones (Low-Beer, T. S., et al., Lancet (1978) 2:1063-65

[0011] Colorectal cancer (Ochsenkuhn, T. et al., American Cancer Society(1999) 1664-69; Hylemon P., Journal of Lipid Research (1997); BernsteinC. et al., Cancer Research (1999) 59, 2353-2357.)

[0012] Barrett's esophaghus and erosive esophagitis (Nehre D et al.,Gut, (1999) 44(5) 598-602; Kauer et al., Surgery, (1997) 122(5) 874-81)

[0013] Arteriosclerosis caused by the presence of Oxidized LDL (FuhrmanB et al., Free Radic Biol Med 34-461997).

[0014] Inflammatory conditions promoted by the presence of COX-2 (Zhanget al., J. Biol Chem (1998) 273(4):2424-8).

[0015] Clearly, improved compounds such as MIPS are required with bothhigher and more specific binding capacity to these particular bile acidsand bile salts. Furthermore, such MIPs would be useful for the treatmentand/or prevention of diseases which are at least partially caused by, orotherwise related to, these specific bile acids and/or salts.Unfortunately, such MIPs are not currently available.

[0016] Illustrative Example

SUMMARY OF THE INVENTION

[0017] The background art neither teaches nor suggests MIPs with bothhigher and more specific binding capacity for particular bile acidsand/or salts, for the treatment and/or prevention of diseases which areat least partially caused by, or otherwise related to, specific bileacids and/or salts.

[0018] The present invention is of improved MIPs with both higher andmore specific binding capacity, including the synthesis of such MIPs,the compounds themselves, and specific applications thereof. The MIPcompounds of the present invention have the advantage of being adaptablefor specific targeted therapeutic and diagnostic uses and/orfunctionality, thereby enabling treatment and diagnosis to be moreeffectively performed.

[0019] As an example of a particularly preferred specific application ofthese compounds, the present invention encompasses the use of MIPs assequestrants in the gastrointestinal tract, particularly in order tobind and therefore remove toxins from the gastrointestinal tract.

[0020] As a particularly preferred example of such toxins, the compoundsof the present invention are useful for the binding and removal ofspecific bile acids and/or salts. Examples of such bile acids and/orsalts include, but are not limited to, a bile acid such as deoxycholicacid (DCA) or the tauro- or glyco-conjugates of DCA. The bile acid orbile conjugate is optionally chenodeoxycholic acid (CDCA) and the glycoor tauro conjugates thereof, or lithocholic acid (LCA) and the glyco ortauro conjugates thereof.

[0021] These bile acids and salts are targeted for removal because oftheir role in various disease states. For example, although bile acidsand salts serve important functions in the body, such as promotingdigestion of fat, more hydrophobic (water-resistant) bile acids, such asDCA, CDCA and LCA, facilitate higher absorption of lipids such ascholesterol and fats into the blood stream and are toxic, causing damageto cells and promoting cancer. Therefore, these more hydrophobic bileacids are highly significant disease-causing agents.

[0022] The novel molecularly imprinted polymers (MIPs) of the presentinvention are able to specifically target and remove significantproportions of these more hydrophobic bile acids. At present there is nodrug that selectively targets the removal of hydrophobic bile acids fromthe body.

[0023] The present invention also encompasses the use of these MIPcompounds for the treatment and/or prevention of diseases which are atleast partially caused by, or otherwise related to, specific bile acidsand/or salts. Examples of such diseases include, but are not limited to,heart disease, particularly by lowering cholesterol levels; treatment ofheart and other oxidized LDL-initiated diseases such as cancer andinflammatory conditions by lowering oxidized LDL; cholesterol-relatedgallstones; colorectal cancer and its precursors; inflammatory diseaseinitiated by the presence of Cox-2, gastro-esophageal reflux diseases(GERD) including Barrett's esophagus, corrosive esophagitis, andesophageal cancer. In addition, the present invention encompasses theuse of such compounds to concurrently treat and/or prevent two or moreof the above diseases.

[0024] The present invention also encompasses use of the MIP medicationin combination with other medications where there is a need for moreeffective treatment which cannot be achieved by the MIP medicationalone. These medications may involve mechanisms of action that lower orchange the composition of bile acids and/or salts in the body or by adifferent mechanism.

[0025] The present invention also encompasses the use of the compoundsof this invention for diagnosis and monitoring of compounds by bindingto a marker and using known binding indicator techniques such asfluorescence. The analysis is performed on samples taken from the body.In a particularly preferred example, the compounds of the presentinvention are useful for the diagnosis and monitoring of specific bileacids and salts. Examples of such bile acids and salts include, but arenot limited to, a bile acid such as deoxycholic acid (DCA) or the tauro-or glyco-conjugates of DCA. The bile acid or bile conjugate isoptionally chenodeoxycholic acid (CDCA) and the glyco or tauroconjugates thereof, or lithocholic acid (LCA) and the glyco or tauroconjugates thereof.

[0026] These bile acids and salts may be targeted for diagnosis andmonitoring because of their roles in various disease states. Forexample, although bile acids serve important functions in the body, suchas promoting digestion of fat, more hydrophobic (water-resistant) bileacids, such as DCA, CDCA and LCA, are toxic, causing damage to cells andpromoting cancer. Therefore, these more hydrophobic bile acids arehighly significant disease-causing agents.

[0027] The novel molecularly imprinted polymers (MIPs) of the presentinvention are able to specifically bind these hydrophobic bile acids,and then to be detected by using known binding indicator techniques suchas fluorescence, for thereby quantifying the levels of the morehydrophobic bile acids.

[0028] The present invention also encompasses the use of these MIPcompounds for the monitoring and diagnosis of diseases which are atleast partially caused by, or otherwise related to, specific bile acidsand/or salts. Examples of such diseases include, but are not limited to,colorectal cancer and its precursors; and esophageal diseases includingBarrett's esophagus, erosive esophagitis, and esophageal cancer bymonitoring the levels of at least one bile acid or salts or the ratiosof at least one bile acid or salts to other bile acids and/or salts.

[0029] The present invention solves a number of long-felt needs. Thereis a need for more effective medication, for example, with patients whodo not respond to conventional treatment, and/or have severe conditionsfor which the currently available medication is insufficient for therequired treatment. This may be achieved with the use of MIP therapy,alone or optionally in combination with other medication. The use ofcombination therapy is well established. These optional, additionalmedications may operate according to mechanisms of action that lower orchange the composition of bile acids and/or salts in the body or by adifferent mechanism.

[0030] There is the need for simple screening techniques for diagnosisand monitoring, especially for asymptomatic conditions. An illustrativeexample is treatment of cancer where the earlier diagnosis of the canceror pre-cancerous condition can significantly impact on the success oftreatment and also contribute to preventing the condition. In some casesthere is need for improving and simplifying existing methods; in othersno existing method exists. MIPs have been used for the diagnosis andmonitoring of various diseases by selectively binding to an establishedmarker which is then identified using known binding indicator techniquessuch as fluorescence. In many cases there will be a need for theimproved MIPs to achieve the required sensitivity.

[0031] As an example for implementation, the present invention isdescribed with regard to the use of MIPs for diagnosis of medicalconditions which are related to, and or characterized by, an effect ofbile acids and /or bile salts, such as atherosclerosis, various diseasesof the gastrointestinal tract, cancer and inflammatory conditions. Thisis achieved by determining the level of at least one specific bile acidor the ratio of at least one specific bile acid to at least a secondspecific bile acid and determining whether these levels fall within anestablish range which indicates the potential existence of the relevantdisease. The analysis is performed on bile acids and or bile salts foundin serum, bile, gastric contents, and feces.

[0032] Hereinafter, the term “treatment” or “treat” for a disease statealso includes the prevention of the occurrence of one or more symptomsand/or effects of the disease state itself. Hereinafter, the term “bileacid” includes both conjugated and unconjugated bile acids unlessotherwise indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

[0034]FIGS. 1A and 1B are schematic descriptions of methods for makingand using the compounds of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention is of improved MIPs with both higher andmore specific binding capacity, including the synthesis of such MIPs,the compounds themselves, and specific applications thereof. Inaddition, the present invention is also useful for treatment of variousdiseases which are related to, and/or characterized by an effect of,bile acids, such as atherosclerosis, liver disease and various diseasesof the gastrointestinal tract. The MIP compounds of the presentinvention are also useful for combination therapy with othermedications.

[0036] The compounds of the present invention are improved MIPs.According to a preferred embodiment of the present invention, thecompound comprises a molecularly imprinted polymer featuring at leastone functional monomer selected from the group consisting of: 2-, 3- and4-vinyl-2-hydroxypyridine; the family of functional monomers4-vinylbenzamide and N-methyl (and other N-alkyl) derivatives, i.e.,N-alkyl-(4-vinylbenzamide) derivatives, and N,N-dimethyl-, N-methyl,N-alkyl-, and N,N-dialkyl-(4-vinylbenzamide) derivatives. Preferably,the functional monomers are cross-linked with at least one crosslinkingagent which is an N,N′-(4-vinylbenzoyl)-1, ω-diaminoalkane. According toother preferred embodiments of the present invention, the molecularlyimprinted polymer features at least one functional monomer of DEVPA(N,N′-diethyl(4-vinylphenyl)amidine). More preferably, the molecularlyimprinted polymer features at least one additional functional monomer,including acrylamide, methacrylamide, N-methylacrylamide,N-methylmethacrylamide, and related derivatives or analogs of thesesubstances. Most preferably, the crosslinking agent for linking thefunctional monomers is ethyleneglycol dimethacrylate, N,N′-diacryloyl-or N,N′-dimethacryloyl ethylenediamine or mixtures of these and relatedderivatives or analogs of these substances.

[0037] Alternatively, the crosslinking agent is selected from the groupconsisting of N,N′-diacryloyl- or N,N′-dimethacryloyl1,3-diaminobenzene, N,N′-diacryloyl- or N,N′-dimethacryloyl1,4-diaminobenzene, the diacrylates or dimethacrylates of 1,2-, 1,3-, or1,4-dihydroxybenzene as well as N,N′-(4-vinylbenzoyl)-1,ω-diaminoalkane.

[0038] Also alternatively, the crosslinking agent is selected from thegroup consisting of an N,N′-diacryloyl- or N,N′-dimethacryloyl1ω-diaminoalkane.

[0039] As an example of a particularly preferred specific application ofthese compounds, the present invention encompasses the use of the MIPsas sequestrants in the gastrointestinal tract, particularly in order tobind and therefore remove toxins from the gastrointestinal tract.

[0040] As a particularly preferred example of such toxins, the compoundsof the present invention are useful for the binding and removal ofspecific bile acids and/or salts. Examples of such bile acids and/orsalts include, but are not limited to, a bile acid such as deoxycholicacid (DCA) or the tauro- or glyco-conjugates of DCA, but may optionallybe chenodeoxycholic acid and the glyco or tauro conjugates thereof, orlithocholic acid and the glyco or tauro conjugates thereof.

[0041] The present invention encompasses the use of these compounds forthe treatment and/or prevention of diseases which are at least partiallycaused by, or otherwise related to, specific bile acids and/or salts.Examples of such diseases include, but are not limited to, heartdisease, particularly by lowering cholesterol levels; treatment of heartand other oxidized LDL-initiated diseases such as cancer andinflammatory conditions by lowering oxidized LDL; cholesterol-relatedgallstones; colorectal cancer and its precursors; and esophagealdiseases including Barrett's esophagus, erosive esophagitis, andesophageal cancer and its precursors. In addition, the present inventionencompasses the use of such compounds to concurrently treat and/orprevent two or more of the above diseases.

EXAMPLE 1

[0042] Method for Synthesis of the Compounds of the Present Invention

[0043] The following description is intended as a non-limiting exampleof methods for synthesizing the compounds of the present invention, byforming the molecularly imprinted polymers from the selected functionalmonomer groups.

[0044] Monomer Synthesis and Characterization.

[0045] A large variety of functional monomers were synthesized in orderto be able to choose a mix of substances that would afford optimalperformance in bile acid and bile salt binding. These included DEVPA(N,N′-diethyl(4-vinylphenyl)amidine) [G. Wulff, T. Gross, R. SchoenfeldAngew. Chem., Int. Ed. Engl. 1997, 36, 1962;. G. Wulff, R. Schoenfeldt,M. Gruen, R. Baumstark, G. Wildburg, L. Haussling, German Patent, BASFAktiengesellschaft, 9610, O. Z. 0050/46933, 1994], and the commonly usedfunctional monomers including methyl methacrylate, acrylamide,methacrylamide, N-methylacrylamide, N-methyl-methacrylamide, and relatedderivatives or analogs of these substances as described in theabove-mentioned reviews on molecularly imprinted polymers. Thefunctional monomers 2-, 3- and 4-vinyl-2-hydroxypyridine were also used.The family of functional monomers 4-vinylbenzamide and N-methyl (andother N-alkyl) derivatives, i.e., N-alkyl-(4-vinylbenzamide)derivatives, and N,N-dimethyl-, N-methyl, N-alkyl-, andN,N-dialkyl-(4-vinylbenzamide) derivatives were also used. The bindingconstants of various functional monomers with bile acids and bile saltswere determined using published methods. Thus, the binding constant forcomplexation of DEVPA and glycodeoxycholic acid was established asKa=1.4×10³ M⁻¹, in DMSO-d₆ at 16° C., using the single point NMR method[D. A. Stauffer, R. E. Barrans, Jr., and D. A. Dougherty, J. Org. Chem.,55, 2762-2767 (1990)].

[0046] Polymer synthesis. The MIPs were prepared using methods in theliterature as referenced above and modifications of these methods. Themethods included bulk polymerization and suspension polymerization withheating and with irradiation initiation using AIBN and relatedinitiators of polymerization. For both bulk polymerization andsuspension polymerization at least two functional monomers were used inaddition to at least one crosslinker. Bulk polymerization proceededusing DMF, ethanol, ethanol-acetonitrile, acetonitrile, DMSO, and mixesof these solvents as the porogen. The polymerization solutions ormixtures were degassed using three freeze-evacuate-thaw cycles andflame-sealed in glass tubes. The suspension polymerization followedmethods used as previously published (Svec, et. al., Angew. Macromol.Chem., 7, 135 (1975). Typically, a dispersion phase of 0.1 to 1-wt. %polyvinyl pyrolidone, 0.2 to 2-wt % polyvinyl alcohol in water, and anorganic phase consisting of the bile acid or salt, monomers andcrosslinker(s) in octanol/cyclohexanol mixtures.

[0047] Table 1 presents illustrative examples of MIPs prepared usingbile acids and bile salts as the template/imprint molecules. TABLE 1Composition and characterization of MIPs^(a) Surface Pore Mean poreTemplate area volume radius MIP Template Porogen removal (m²/g) (ml/g)Porosity (nm) P1 GDCA MeCN + EtOH   94% 116 1.243 61.8% 21.4 (1:1 v/v)P2 CDCA EtOH   90% 129 1.437 65.1% 22.3 P3 LCA THF 77+% 145 1.094 58.7%15.1 # the template was removed by treating with 1 M NaOH to obtain thecorresponding MIPs.

[0048] Binding studies. The binding experiments were performed usingliterature procedures for related substances [M. J. Whitcombe, M. E.Rodrigues, P. Villan, E. N. Vulfson, J. Am. Chem. Soc., 117, 7105-7111(1995)]. The dried polymers (1-40 mg) were weighed into 2-ml screw-capvials and 1 ml of a 50% aqueous ethanolic ligand solution (50 iM) wereadded to the vials which were then placed in a sample shaker overnight.

[0049] For analysis of bile acids, the polymer suspensions were verycautiously filtered directly into fresh screw-cap vials containing 3-mlof conc. sulfuric acid, cooled in ice. The solutions were maintained at80° C. for one hour and then cooled to room temperature. They were thenanalyzed by UV-VIS spectroscopy using a UVIKON-930 instrument accordingto a reported method [A. Fini and P. Zuman, Collect. Czech. Chem.Commun. 58, 53 (1993)]. For each bile acid and bile salt a selectedwavelength was used and the ligand concentration was determined byreference to a calibration curve which was prepared using five samplesof known concentration. All determinations were performed in duplicate.The wavelengths used for each analyte was as follows: deoxycholic acid,385 nm; glycodeoxycholic acid and tauro deoxycholic acid, 309 nm;chenodeoxycholic acid, 382 nm; lithocholic acid, 308 nm.

[0050] A more specific, non-limiting example is described with regard toCDCA. CDCA-binding polymer MS39 (capacity=187 micromol/g=1.47 mg ofCDCA/20 mg of MS39) was allowed to stand in aqueous ethanolic solutionsof CDCA for 20 hrs, containing various levels of CDCA; it was previouslyshown that this time was sufficient for attaining equilibrium binding.The analyses of CDCA were achieved using the above-mentionedspectroscopic method (A. Fini and P. Zuman, 1993). In this way, thebinding capacity and calibration curves and specificity of theCDCA-binding polymer MS39 were determined. Thus, in one of the aboveexperiments, MS39 absorbed 1.15±0.04 mg of CDCA while a control polymerabsorbed 0.17±0.05 mg of CDCA under identical conditions.

[0051] For diagnostic assays, CDCA-binding polymer was charged withtritium-labelled CDCA. The release of radiolabeled CDCA was monitoredunder specified conditions and also in the presence of varying levels of“cold” CDCA. In this way, it was possible to accurately estimate CDCA inunknown samples. In a modification of this method, the MIP was preparedas a thin layer in the wells of microtiter plates; the plates wereincubated with tritium-labelled CDCA, washed, and then used to preparecalibration curves and for the assay of CDCA.

[0052] In a different modification of the above, a fluorescentderivative of cholic acid, in which the 12-alpha position wassubstituted with a fluorophore such as coumarin, was used as the assaysubstance. CDCA-binding MIPs were charged with this fluorescent-labelledCDCA-analog and used to develop both a microplate and a free MIPparticle assay for CDCA.

EXAMPLE 2

[0053] Methods and Compositions for Administration

[0054] The compounds of the present invention can be administered to asubject by various ways, which are well known in the art. Hereinafter,the term “subject” refers to the human or lower animal to whom thetherapeutic agent is administered. For example, administration may bedone topically (including ophthalmically, vaginally, rectally,intranasally and by inhalation), orally, or parenterally, for example byintravenous drip or intraperitoneal, subcutaneous, or intramuscularinjection.

[0055] Formulations for topical administration may include but are notlimited to lotions, ointments, gels, creams, suppositories, drops,liquids, sprays and powders. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be necessaryor desirable.

[0056] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets,capsules or tablets. Thickeners, diluents, flavorings, dispersing aids,emulsifiers or binders may be desirable.

[0057] Formulations for parenteral administration may include but arenot limited to sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

[0058] Dosing is dependent on the severity of the symptoms and on theresponsiveness of the subject to the compound of the present invention.Persons of ordinary skill in the art can easily determine optimumdosages, dosing methodologies and repetition rates.

EXAMPLE 3

[0059] Method of Treatment of Bile Salt or Acid-Related Conditions

[0060] As noted above, the compounds of the present invention arebelieved to be useful for the treatment of disease states which arerelated to bile acids, by binding to and thereby removing the bile acidfrom the gastrointestinal tract of the subject. The following example isan illustration only of a method of treating such a condition with thecompound of the present invention, and is not intended to be limiting.

[0061] The method includes the step of administering a compound, in apharmaceutically acceptable carrier as described in Example 2 above, toa subject to be treated. The compound is administered according to aneffective dosing methodology, preferably until a predefined endpoint isreached, such as the absence of a symptom of the disease condition inthe subject, or the prevention of the appearance of such a symptom inthe subject.

[0062] Examples of such diseases, disease conditions or disease statesinclude, but are not limited to, heart disease, treatment of heart andother oxidized LDL-initiated diseases such as cancer and inflammatoryconditions, cholesterol gallstone, colorectal cancer and its precursors,esophagus diseases including Barrett's esophagus, erosive esophagitisesophageal cancer and its precursors and COX-2 mediated inflammatoryconditions.. These disease conditions are described in more detail inthe subsections below.

[0063] Reduction of Cholesterol Levels

[0064] There is a well-established relationship between cholesterollowering and reduction in atherosclerosis and heart disease. Over 6million patients in the US use cholesterol-lowering drugs and it isestimated that 25 million people in the US should be takingcholesterol-lowering drugs. Statins (i.e., inhibitors of HMG CoAreductase, the enzyme required for the body's production of cholesterol)are the main class of drug used.

[0065] Bile sequestrants have been used for many years as the firstlevel drug treatment for cholesterol reduction. Its use has beenlimited, and is even decreasing, because it is inconvenient to use,requiring high dosages and not palatable. Geltex Pharmaceuticals Inc.(F.D.A. N.D.A 21-141, N.D.A. 21-176) has sought to overcome thesedisadvantages and obtained FDA approval for new polymeric bilesequestrants. However, these still require significant dosages and otherdesirable nutrients may be removed.

[0066] The MIPs provide a convenient and selective bile-sequestrantwhich, unlike existing polymeric agents, do not remove desirablesubstances (e.g., vitamins and medications). Another advantage is thatremoving the more hydrophobic bile acids will have a greater impact onreducing cholesterol than the removal of other bile acids because thehydrophobic bile acids promote the presence of cholesterol in the blood(Reno Z. et al. in Gastroenterology Clinics of North America, ed. CooperA. D., W. B. Saunders Company (1999) p.1; Cooper A. D., inGastroenterology Clinics of North America, ed. Cooper A. D. W. B.Saunders Company (1999) p. 211).

[0067] Particular cholesterol lowering applications where the specificbile sequestrant MIPs will be preferred include, but are not limited to,the following examples. First, for patients with high risk ofcholesterol and the other medical indications where the removal of themore hydrophobic bile acids is warranted, the MIP compounds of thepresent invention are preferred. Examples include high-risk colon cancerpatients, patients with Barrett's esophagus and at a high risk ofgallstones due to dieting. Other suitable candidates include youngpatients who need to take cholesterol lowering drugs long-term to avoidpotential damage to the liver; and patients requiring combinationtherapy with statins, for example, patients with very high cholesterollevels.

[0068] Reduction of Oxidized LDL

[0069] In the past decade it has been recognized that oxidized LDL andnot LDL is the real culprit in heart disease. It damages the lining ofblood vessels, causes growth of cells that form the wall of the bloodvessel and causes thickening and narrowing of the blood vessel—all ofwhich are recognized to contribute to heart attacks and strokes. Thus,there is a need to reduce the oxidation of LDL.

[0070] In vitro research has demonstrated that DCA can induce theconversion of LDL to oxidized LDL. Bile acids are exposed to LDL whenthey circulate in the blood (Ljubuncic P. et al., Gut (1996); 39:475-8).

[0071] From the intestine, bile acids enter into the portal vascularsystem where they are associated with plasma and lipoproteins.Biophysical properties of primary or secondary bile acids dictatewhether they associate with lipoproteins or albumin as is most often thecase or if they are freely soluble in plasma. Approx. 10%-30% of bileacids in the portal system escape subsequent extraction by the liver andspill over into the systemic circulation. As a result the blood vesselsof the heart are exposed to DCA in the bile acids. The reduction of DCAthen is used to reduce the level of oxidized LDL.

[0072] The MIP compounds of the present invention may be preferred as acombined cholesterol and oxidized LDL lowering medication or incombination with statins as the mechanism of action is different.

[0073] Treatment of GERD & Esophagus Cancer

[0074] Gastro-esophageal Reflux Disease (GERD) refers to a range ofsymptoms that result from the exposure of the esophagus to gastric acid.Heartburn and regurgitation are the most common symptoms. Approximately10% of the American population experience heartburn daily. Although GERDis a manageable disease it can evolve into erosive esophagitis wherethere is inflammation and tissue damage to the esophagus. If leftuntreated, complications may occur including hemorrhage and Barrett'sesophagus. An estimated and rapidly increasing 1-2 million Americanpatients (average age 55-65) progress to Barrett's esophagus (i.e.,changes in the appearance of the lining of the esophagus). Over thecourse of time, 5-10% of these patients will develop esophagus cancerwhich has a very poor survival rate (i.e., 5% in 5 years). Barrett'sesophagus leads to a 30-40 fold increase in the risk of esophaguscancer, the fastest growing cancer in Western countries.

[0075] Currently, the main medication for severe GERD (esophagitis andBarrett's Esophagus) are drugs that block production of acid and relieveirritated tissue. The most effective drugs are proton pump inhibitorswith weaker histamine H2 receptor antagonists also sometimes prescribed.

[0076] The combination of gastric juice and bile acids in esophaguspatients is considered decisive in the development of many cases ofBarrett's esophagus. Additionally, recent studies have shown that manypatients with erosive esophagus and Barrett's esophagus havesignificantly higher concentrations of the more toxic hydrophobic bileacids and overall bile acid concentrations in the esophagus. In healthypatients this normally is not the case (Nehra D., et al., Gut, (1999);44:598;Kauer W. K. H. et al., Surgery (1997); 122:874-81). Research alsoindicates that exposure to bile acids and in particular DCA and CDCA aredecisive factors contributing to the development cancer (Zhang et al., JBiol Chem (1998); 273:2424-8; Shirvani et al. Gastroenterology 2000;118: 487-496).

[0077] The medication that suppresses gastric juice does not halt thereflux itself. Even if medication renders a GERD patient asymptomatic,this does not always indicate complete control of the reflux andasymptomatic reflux will lead to progression of Barrett's esophagus.Furthermore, the pharmacologically altered gastric contents,particularly bile acids, may contribute to the progression of Barrett'sesophagus. Significantly, the bile appears to be decisive for thedevelopment of adenocarcinoma (Demeester T. R., “Without Bile- NoAdenocarcinoma with Barrett's Esophagus”, Presented at InternationalWorkshop “From Reflux to Barrett's Carcinoma,” Mar. 23-25, 1998,European Surgical Institute). Recent research suggests that a reason forthis effect may be that gastric acid suppression treatment leads tobacterial overgrowth including species that deconjugate bile. This leadsto a significant increase in the concentration of unconjugated bileacids (Shido K et al., Gut 1998 42(2): 266-71; Thieson J. et al., JGastointest Surg 2000 4(1): 50-4).

[0078] As a result of the limitations of existing medical treatment withacid suppressants, anti-reflux surgery is being considered. It also hasnot proven effective in many cases to lead to the regression ofBarrett's esophagus and the risk of cancer remains. It is consideredcontroversial whether surgery should be performed on all individualswith Barrett's esophagus especially those whose symptoms are wellcontrolled with medication. Photodynamic therapy also is being used inclinical trials in an effort to remove the lesions and anti-cancerousmedication are being examined for treating the Barrett's esophaguslesions. However they do not treat the cause and there are concerns thatthese approaches may only partially prevent the progression to cancer.

[0079] Accordingly, there is a need for alternative treatments. Thespecific bile acid binding MIPs offer an alternative medical approachfor treatment of severe GERD and Barrett's esophagus. Removal of themost toxic bile acids in the gastrointestinal tract before they enterthe esophagus should limit the development of severe GERD and Barrett'sesophagus and assist in the healing process. Combination therapy withthe gastric juice suppressants may be particularly effective inpreventing Barrett's esophagus with asymptomatic patients who may stillsuffer from reflux and in overcoming the potential for the proton pumpinhibitors contributing to the progression of Barrett's esophagus.

[0080] Furthermore, bile acids have been found at higher pH levels withunconjugated bile acids (e.g., DCA) being most gastrotoxic under theseconditions. There are significant numbers of non-responders to thegastric acid inhibitor medication and the MIPs may be used to treatnon-responders where the reason is the presence of bile acids at thehigher pH levels.

[0081] Since these bile acids are decisive in the development ofadenocarcinoma, the specific bile acid binding MIPs may prove effectivein promoting the regression of esophagus lesions. They may prove veryeffective in combination with the photodynamic therapy-based cancerousand pre-cancerous killing agents being developed for use to treatBarrett's esophagus.

[0082] Treatment of Colon Cancer

[0083] Colorectal cancer is one of the most lethal cancers. In the USAit is the number two killer cancer with nearly 55,000 deaths and 140,000new cases annually. High risk groups include those with a family historyof polyps, over the age of 50, patients who have had colon polypsremoved and who have had colon cancer (over 1.2 million people in theUS).

[0084] Colorectal cancer provides unique opportunities for primaryintervention among human malignancies because it progresses throughclinically recognizable stages from normal mucosa through enlarging andincreasingly dysplastic polyps which eventuate in carcinoma.

[0085] The precursor relationship of colorectal adenoma to carcinoma andthe high prevalence of adenomas make them an attractive target inchemoprevention. The prevalence of polyps increases with age in moderateand high-risk populations, reaching 20-40% at the age of 50-60 years,and 50% or more for individuals older than 70 years. The steepestincrease in adenoma prevalence occurs between the ages of 50-59.However, removal of polyps does not change the pathogenetic milieuresponsible for their growth and development. The recurrence rate forcolorectal adenomas has been variably reported, but most studiesdocument an adenoma recurrence rate of 20-60% after two years. Forpatients who had newly diagnosed adenomas removed during colonoscopy,28% had additional polyps detected at a one-year follow-up examination,and of those patients, 22% had new adenomatous polyps again detected onexamination two years later. Patients who have undergone surgicalresection of a primary colorectal cancer have also been shown to be athigh risk of developing metachronous adenomas. Thus, there is a need foradditional treatment to prevent the recurrence of the polyps.

[0086] The cyclooxygenase-2 (COX-2) inhibitor, Celebrex, has beenapproved for adjuvant treatment of colon polyps. However, the highestreported reduction rate has been 28% (FDA approved labeling). Thus,there remains the need for improved treatment. Other approaches arerevealed in Larson et al. (U.S. Pat. No. 5,945,411) whereursodeoxycholic acid is used in combination with sulindac and phenolicNSAID to prevent the recurrence of colorectal adenomas followingremoval. However the ursodeoxycholic acid is not selective and theamount of the more toxic bile it removes is limited.

[0087] Recent research with more precise sampling supports a connectionbetween DCA and colorectal cancer (Bayerdorffer E. et al., Gut (1995);36: 268-273; Ochsenkuhn T. et al., Cancer (1999), 85: 1664-1669).Although only a small fraction of bile acids in the enterohepaticcirculation enter the colon; perturbations of the cycling rate of bileacids by diet (e.g., fat), mucosal dysfunction such as Crohn's diseaseor surgery (e.g., cholecystectomy) may increase the fecal bile acid loador reduce bile acid uptake and cause spillage into the colon. Thus, thereduction of DCA and its removal from the colon can be used as analternative preventive of the pre-cancerous polyps. The MIPs also can beused in combination with other treatments such as Celebrex medication.

[0088] Research also suggests that DCA may increase the proliferationrate of colon cancer cells. Thus, DCA removal may also be a usefulsupplemental treatment for treating colon cancer (Peiffer L P. et al.,Dig Dis Sci, (1997) 42(11):2234-40), This can be alone or in combinationwith other colon cancer medications such as Celebrex or specific canceror pre-cancerous killing agents such as the compounds being developed byCell Pathways Inc.

[0089] Treatment of Liver Disease

[0090] Cholestatic diseases are a group of diseases characterized by theprogressive retention of bile acids in the liver, resulting in liverinjury, cirrhosis and ultimately death. In the USA, primary biliarycirrhosis (PBC) occurs in about 10,000-40,000 patients and primarysclerosing cholangitis (PSC) in about 2,500-16,000 patients. Othercholestatic conditions include: complications of liver or bone marrowtransplantations, complications of cystic fibrosis, neonatal cholestaticsyndromes, cholestasis of pregnancy, autoimmune cholangiopathy, as wellas a variety of drug reactions.

[0091] Ursodeoxycholic acid (UDCA) slows the rate of diseaseprogression, lessens the morbidity and mortality risk and improvesquality of life. In PBC, UDCA can postpone the need for livertransplantation. The probable mechanism of action of UDCA in PBC mayreflect the observation that UDCA changes the amount of toxichydrophobic and non-toxic hydrophilic bile acids in the bile salt poolin favor of nontoxic bile. When the bile cannuliculae are damaged by anautoimmune or other disease process inherent to any of the cholestaticdiseases, bile salts leak into the hepatic parenchyma. The resultinginflammation and the extent of bile salt induced injury is a directresult of the concentration of toxic hydrophobic bile salts present inthe extravasated bile (Oude Elferink R. P. J. and Groen, A. K. inGastroenterology Clinics of North America, Vol 28, ed. Cooper A. D., W.B. Saunders Company (1999) pp.59-74; Salen G. and Batta, A. K.Gastroenterology Clinics of North America, Vol 28, ed. Cooper A. D., W.B. Saunders Company (1999) pp.173-193; Stiehl A., Benz, C. and Sauer, P.Gastroenterology Clinics of North America, Vol 28, ed. Cooper A. D., W.B. Saunders Company (1999) pp.195-209).

[0092] Many patients with cholestatic liver diseases do not respond toUCDA therapy. This includes patients with advanced disease and somefamilial intrahepatic cholestasis patients. Lack of response may reflectan upper limit of how much UDCA can be absorbed so that approximatelyhalf of the UDCA administered is absorbed under the best ofcircumstances.

[0093] Generalized pruritus (itchiness) is a very troublesome symptom inchronic cholestasis. Many patients with pruritus have become suicidalfrom their discomfort. Although incompletely understood, thepathogenesis is believed to be related to the accumulation of toxic bileacids in the skin. Combined UDCA treatment with bile acid sequestrantsactive in the intestine is a partially successful treatment. Bythemselves, bile acid sequestrants are not very effective, probably fora number of reasons.

[0094] For example, bile acid sequestrants do not differentiate betweenthe toxic hydrophobic and nontoxic hydrophilic bile acids such as UCDA.As they are non-specific, when given with UDCA, they absorb the UDCAreducing the effectiveness of this non-toxic bile salt treatment.

[0095] They do not decrease the overall level of bile acids becauseadditional bile acids (toxic and nontoxic) will be produced. The liverregulates the amount of bile salts in the body. As the concentration ofone relative to the other changes, the overall amount of bile saltsremains the same. Thus, a treatment that indiscriminately sequestersbile salts only serves to stimulate more production of both toxic andnon-toxic bile salt species.

[0096] These bile acid sequestrants also bind fat-soluble vitamins suchas vitamin D, K, and E and hydrophobic medications. The long-term use ofthese sequestrants can lead to vitamin deficiencies such as bone diseaseand clotting abnormalities. They also may cause variable delivery ofmedication with the potential for under dosing.

[0097] Thus there is a need for alternative treatment. The MIP compoundsof the present invention offer alternative improved treatment used aloneto remove the more toxic bile acids and/or salts or as combinationtherapy with UDCA for improved treatment of pruritus, when the UDCAalone is not effective, and to improve overall prognosis in conditionsthat warrant UDCA therapy. The MIPs are selective for the toxic bileacids and overcome these problems for a number of reasons.

[0098] First, the MIP compounds of the present invention do not bindUDCA in significant levels. Theoretically, orally administered UCDAabsorption will not be affected by our MIPs and the proportion of totalbile salts that are UCDA will increase in bile, making extrabiliary bileboth less hydrophobic and less toxic.

[0099] In addition, it is possible to target the most hydrophobic andtoxic bile acids, thereby changing the balance between toxic andnontoxic bile acids and thus reducing the overall toxic levels. As theMIPs remove the toxic bile salts, the proportion of total bile that isUCDA will increase. Since the liver attempts to keep the pool of bilesalts constant and orally administered UCDA is replacing the removedtoxic bile salts, the liver will not be stimulated to replenish the bilesalt pool with toxic bile salts.

[0100] In addition, because the compounds of the present invention donot bind other fat-soluble substances such as vitamin D, E, and K orother hydrophobic medications, the potential for hypovitaminosis orundermedication as a consequence of their use is low.

[0101] Treatment of Cholesterol Gallstones

[0102] Approximately 10-15% of the adult population have gallstones. Inthe USA more than 20 million have gallstones with about one-thirddeveloping symptoms. The prevalence is higher in women, obesity (rapidweight loss) as well as in older patients and certain ethnic groups.Gallstones are the most common and costly disease in the USA with anestimated overall cost of more than $5 billion with over 500,000-600,000people undergoing surgery to remove the gallbladder. Most gallstones arecomposed principally of cholesterol.

[0103] Typical treatment for patients with symptoms requiring treatmentis surgical removal of the gallbladder unless they are unable totolerate general anesthesia or have a condition that precludes surgery.Drawbacks of the surgery include recurring pain and possiblecontribution to gallbladder and colon cancer.

[0104] Oral dissolution therapy with UDCA is most effective in 15% ofsymptomatic patients (small non-calcified floating cholesterol stones ofless than 5 mms have 90% chance of dissolution and stones less than 10mms have 60% chance of dissolution). Six to twelve months of therapy arerequired in many patients and monitoring is necessary until dissolutionis achieved.

[0105] This treatment is used when operation is not possible and forpatients who choose to avoid operation or for prophylactic treatment forspecial groups, e.g., diabetic patients, sickle cell patients, childrenpre-transplantation and/or immuno-suppressed patients and for patientsundergoing rapid weight loss. The main drawback is the recurrence ofgallstones (in about half of those treated stones return within 5years).

[0106] Deoxycholic acid is implicated in gallstone disease. Cholesterolhypersecretion triggers events that lead to loss of gallbladderreservoir function leading to increased degradation of primary bilesalts to secondary bile salts (such as DCA) by the bacteria in theintestine. This leads to an increased proportion of DCA which enhancescholesterol hypersecretion into bile and also accelerates cholesterolcrystallization (Beer F. et al, J Clin Invest (1992); 90:859-868; ShodaJ. et al., Hepatology (1995); 21:1291-1302).

[0107] It has been found that after gallstone patients were treated withantibiotics that kill the bacteria converting cholic acid to DCA, theirDCA levels and cholesterol supersaturated bile dropped below thethreshold required to develop gallstones. However, broad-scaleantibiotics are not practical as a treatment because they tend to wipeout both “good” and “bad” bacteria in the intestine.

[0108] The reduction of DCA by the MIP compounds of the presentinvention, in combination with the administration of UDCA, are effectiveas an alternative oral treatment to reduce cholesterol supersaturatedbile, to shorten the time required for dissolution, to be more effectivefor larger stones and reduce the risk of recurrence.

[0109] Anti-Inflammatory Effects

[0110] The cyclooxygenase-2 (COX-2) inhibitors have been shown toexhibit anti-inflammatory effects. Research shows that DCA and CDCApromote the expression of COX-2 (Zhang et al., J Biol Chem (1998);273:2424-8; Shirvani et al. Gastroenterology 2000; 118: 487-496).

[0111] From the intestine, bile acids enter into the portal vascularsystem where they are associated with plasma and lipoproteins.Biophysical properties of primary or secondary bile acids dictatewhether they associate with lipoproteins or albumin as is most often thecase or if they are freely soluble in plasma. Approx. 10%-30% of bileacids in the portal system escape subsequent extraction by the liver andspill over into the systemic circulation. The bile that circulates inthe blood can enhance the expression of COX-2 production so that itsreduction will have an anti-inflammatory effect, for example, intreating rheumatoid arthritis.

[0112] The MIP compounds of the present invention could be used alone orin combination with other anti-inflammatory drugs such as Celebrex.

[0113] In addition to using MIPs to sequester and remove bile acids, theuse of MIPs is described, in combination with other non-specificmedications which can reduce the level of the more toxic bile acids,such as non-MIP polymer bile sequestrants (e.g., colesevelamhydrochloride developed by Geltex Pharmaceuticals Inc.), UDCA or itsderivatives which replace the more toxic bile acids. Additionally, theMIPs may be used with antibiotic or other agents to prevent theconversion of the relatively harmless cholic acid to the toxicdeoxycholic acid by 7-alpha-dehydroxylating bacteria (Berr F. et al.Gastroenterology (1996) 111:1611-1620).

[0114] Alternatively, these agents that can reduce the levels of themore toxic bile may be used to treat new medical indications includingadvanced GERD (esophagitis, Barret's esophagus), esophagus cancer,oxidized LDL for atherosclerosis and anti-inflammatory treatment.

EXAMPLE 4

[0115] Method of Treatment of Bile Salt or Acid-Related Conditions UsingMIPs in Combination with Other Medication

[0116] As noted above, the compounds of the present invention may not besufficiently effective for the treatment of disease states which arerelated to bile acids, by binding to and thereby removing the bile acidfrom the gastrointestinal tract of the subject. In order to achieve thedesired treatment, the compounds of the present invention may be used incombination with other medications. The medications may involvemechanisms of action that lower or change the composition of bile acidsand/or salts in the body or act by a different mechanism. It isimportant to note that since the MIPs are selective, there is only avery limited possibility that the MIPs will bind with the combinationtherapy medication, as has occurred for example when non-specific bilesequestrants are used in combination therapy causing in some casesvariable delivery of medication with the potential for under-dosing.

[0117] The following example is an illustration only of a method oftreating such a condition with the compound of the present invention,and is not intended to be limiting.

[0118] The method includes the step of administering a MIP compound, ina pharmaceutically acceptable carrier as described in Example 2 above,together with another medication to a subject to be treated. Thecompounds are administered according to an effective dosing methodology,for example together or separately, and preferably until a predefinedendpoint is reached, such as the absence of a symptom of the diseasecondition in the subject, or the prevention of the appearance of such asymptom in the subject.

[0119] Illustrative but not limiting examples of medication for alteringthe level or composition of bile acids in the body include thefollowing.

[0120] Bile sequestrants have been used for many years as the firstlevel drug treatment for cholesterol reduction. Its use has been limitedand is even reducing because it is very inconvenient to use, requiringvery high dosages and is not palatable. Geltex Pharmaceuticals Inc.(F.D.A. N.D.A 21-141, N.D.A. 21-176) has sought to overcome thesedisadvantages and obtained FDA approval for new polymeric bilesequestrants. However, these still require significant dosages and otherdesirable nutrients may be removed.

[0121] These bile sequestrants have been used before in combinationtherapy with ursodeoxycholic acid (UDCA) for treatment of livercholestatic diseases but the limitations particularly there ability tobind the UDCA has limited their effectiveness (Oude Elferink R. P. J.and Groen, A. K. in Gastroenterology Clinics of North America, Vol 28,ed. Cooper A. D., W. B. Saunders Company (1999) pp.59-74.Salen G. andBatta, A. K. Gastroenterology Clinics of North America, Vol 28, ed.Cooper A. D., W. B. Saunders Company (1999) pp.173-193. Stiehl A., Benz,C. and Sauer, P. Gastroenterology Clinics of North America, Vol 28, ed.Cooper A. D., W. B. Saunders Company (1999) pp.195-209. Cooper A. D., inGastroenterology Clinics of North America ed. Cooper A. D. W. B.Saunders Company (1999) p. 211).

[0122] Ursodeoxycholic acid (UDCA) is a non-toxic bile acid which isalso used for the treatment of gallstones and primary biliary cirrhosis.It has been used with limited success in combination therapy withnon-specific bile sequestrants for primary biliary cirrhosis, especiallyfor the treatment of pruritis. (Oude Elferink R. P. J. and Groen, A. K.in Gastroenterology Clinics of North America, Vol 28, ed. Cooper A. D.,W. B. Saunders Company (1999) pp.59-74.Salen G. and Batta, A. K.Gastroenterology Clinics of North America, Vol 28, ed. Cooper A. D., W.B. Saunders Company (1999) pp.173-193. Stiehl A., Benz, C. and Sauer, P.Gastroenterology Clinics of North America, Vol 28, ed. Cooper A. D., W.B. Saunders Company (1999) pp.195-209. Cooper A. D., in GastroenterologyClinics of North America ed. Cooper A. D. W. B. Saunders Company (1999)p. 211).

[0123] Bile acids are absorbed from the small intestine by a passive andan active mechanism. Bile acid transport inhibitors are being developedfor treating bile related disease (Glaxo Welcome 264w94 in Phase IIclinical trials and U.S. Pat. No. 5,589,358; Cooper A. D., inGastroenterology Clinics of North America ed. Cooper A. D. W. B.Saunders Company (1999) p. 211). These will have the effect of reducingbile circulated in the body. Antibiotics have been used to kill the 7alpha-dehydroxylating bacteria that enhance deoxycholic acid input byconverting cholic acid to DCA (Beer F. et al Gastroenterology 1996December; 11(6):1611-2), P. Hylemon of Virginia Commonwealth University,in cooperation with and Hoechst Marion Roussel, is developing specificinhibitors of the bacteria in the colon that convert cholic acid to DCA.Other approaches to inhibit these bacteria may also be considered. Theseapproaches also may be used for the bacteria found to convert conjugatedbile to unconjugated bile found for example when acid inhibitormedication is taken for GERD treatment (Thieson J. et al., J GastointestSurg 2000 4(1): 50-4).

[0124] Antibodies may bind bile acids so that their activity isneutralized. Non-limiting examples of antibodies raised against bileacids include those outlined in U.S. Pat. Nos. 5,631,138 and 5,976,811.

[0125] Illustrative but not limiting examples of combination therapy ofMIP with medication for altering the level or composition of bile acidsin the body include the following.

[0126] Treatment of Liver Disease

[0127] As outlined in Example 3 above UDCA is used in treating PBC. Theprobable mechanism of action of UDCA in PBC may reflect the observationthat UDCA changes the amount of toxic hydrophobic and non-toxichydrophilic bile acids in the bile salt pool in favor of nontoxic bile(Oude Elferink R. P. J. and Groen, A. K. in Gastroenterology Clinics ofNorth America, Vol 28, ed. Cooper A. D., W. B. Saunders Company (1999)pp.59-74. Salen G. and Batta, A. K. Gastroenterology Clinics of NorthAmerica, Vol 28, ed. Cooper A. D., W. B. Saunders Company (1999)pp.173-193. Stiehl A., Benz, C. and Sauer, P. Gastroenterology Clinicsof North America, Vol 28, ed. Cooper A. D., W. B. Saunders Company(1999) pp.195-209).

[0128] Also as outlined in example 3, there is a need for alternativetreatment to UDCA. The MIP compounds of the present invention offeralternative improved treatment. However in other cases, use of the MIPsmay be insufficient to remove the required levels of the more toxic bileacids and/or salts to effect the desired treatment. Combination therapyof the MIPs with UDCA may be effective for the improved treatment ofcholestatic disease and pruritus, when the MIP alone is not effective,and to improve overall prognosis in conditions that warrant MIP therapy.

[0129] The MIP compounds of the present invention are ideal forcombination therapy as they do not bind UDCA in significant levels.Orally administered UDCA absorption should not be affected by the MIPcompounds and the proportion of UDCA total bile salts will increase inbile, making the bile both less hydrophobic and less toxic.

[0130] Treatment of Cholesterol Gallstones

[0131] As outlined in Example 3, specific MIPs that bind bile acids,particularly DCA. In severe or other cases, such MIPs may not besufficient to achieve effective treatment especially of largergallstones. The reduction of DCA by the MIP compounds of the presentinvention, in combination with the administration of UDCA, are effectiveas an alternative oral treatment to reduce cholesterol supersaturatedbile, to shorten the time required for dissolution, to be more effectivefor larger stones and to reduce the risk of recurrence.

[0132] Illustrative but not limiting examples of combination therapymedication of MIPs with other medication that acts by a mechanism thatdoes not alter the levels or composition of bile acids include:

[0133] Reduction of Cholesterol Levels

[0134] As outlined in example 3, bile-binding MIPs may be used to lowercholesterol. Where cholesterol levels are high, the MIP therapy alone orother cholesterol lowering therapy such as statin medications may not besufficient. This approach has been applied with nonspecific bilesequestrants with some success (Geltex Pharmaceuticals Inc. F.D.A. N.D.A21-141, N.D.A. 21-176). However, because they are non-specific, thesesequestrants may interact with the statins leading to underdosing andreduced effect of the statins. As the MIPs and statins also havedifferent mechanisms, they can be used together in combination therapy.The specific MIPs are unlikely to interact with the statins which couldlead to underdosing or to reduced effect of the statins.

[0135] Treatment of GERD & Esophagus Cancer

[0136] As outlined in Example 3, the gastric acid inhibitors may notprevent bile acids from refluxing and causing GERD (esophagitis andBarrett's Esophagus) and contributing to the development of cancer. Infact, these medications may potentiate the damaging effects of bileacids by increasing the levels of bile or changing the bile compositionform conjugated to the potentially more toxic unconjugated bile.

[0137] Combination therapy with the gastric juice suppressants may beparticularly effective in preventing Barrett's esophagus withasymptomatic patients who still suffer from reflux, in overcoming thepotential contribution of proton pump inhibitors to the progression ofBarrett's esophagus and for non-responders.

[0138] Since these bile acids are decisive in the development ofadenocarcinoma, the specific bile acid binding MIPs may prove effectivein promoting the regression of esophagus lesions. They may prove veryeffective in combination with the photodynamic therapy-based cancerousand pre-cancerous killing agents such as those being developed by CellPathways Inc. for treating Barrett's esophagus.

[0139] Treatment of Colon Cancer

[0140] As outlined in Example 3 MIPs targeting specific toxic biles areused to treat colon polyps. The use of MIPs alone in severe or othercases may not be sufficient. In such cases, combination therapy of MIPswith other medication for such treatment may be used. Examples includethe cyclooxygenase-2 (COX-2) inhibitor Celebrex, the pre-cancerous cellkilling agents such as being developed by Cell Pathways Inc. andsulindac and phenolic NSAID.

[0141] Similarly the MIPs may be used to remove the DCA and other bileacids that may increase the proliferation rate of colon cancer cells, aswell as supplemental colon cancer treatment in combination with othercolon cancer medications, such as Celebrex, or with specific cancer orpre-cancerous killing agents, such as the compounds being developed byCell Pathways Inc.

EXAMPLE 5

[0142] Diagnosis/Screening of Bile Acids

[0143] The MIP compounds of the present invention have been used forsensitive assays. They have the advantage over other biological assaysof enhanced stability, simple handling and long-life storage. They canbe coated on the plates used for diagnosis, thus reducing handlingduring diagnosis. The MIPs binding the bile acids have the potential toprovide novel and simple-to-handle diagnostic systems for the rapid andsensitive quantification of bile acids in serum, bile, gastric contentsand feces.

[0144] The MIP compounds of the present invention could prove especiallyeffective in screening for diseases where bile acids contribute to thedevelopment and progression of disease and for the monitoring oftreatment.

[0145] Imprinted polymers have already been used successfully assubstitutes for antibodies in immunoassays (Vlatkis G., et al., Nature1993; 361:645-647). The specific bile acid-binding MIP compounds of thepresent invention can also be used in a variety of formats for sensitiveassays for, respectively, DCA, CDCA, and other bile acids and theirsalts.

[0146] A more specific non-limiting example is described regarding useof a CDCA-binding MIP for a diagnostic assay. The CDCA-binding polymerwas charged with tritium-labelled CDCA. The release of radiolabeled CDCAwas monitored under specified conditions and also in the presence ofvarying levels of “cold” CDCA. In this way, it was possible toaccurately estimate CDCA in unknown samples. In a modification of thismethod, the MIP was prepared as a thin layer in the wells of microtiterplates; the plates were incubated with tritium-labelled CDCA, washed,and then used to prepare calibration curves for the assay of CDCA.

[0147] In a different modification of the above, a fluorescentderivative of cholic acid, in which the 12-alpha position wassubstituted with a fluorophore such as coumarin, was used as the assaysubstance. CDCA-binding MIPs were charged with this fluorescent labelledCDCA-analog and used to develop both a microplate and a free MIPparticle assay for CDCA.

[0148] One illustrative, non-limiting example of a method for diagnosisof a bile-related disease in a subject with the MIP compounds is asfollows. First, a sample of serum is obtained from the subject. Next,the sample is contacted with an MIP compound to determine at least arelative amount of at least one bile acid in the tissue sample. Thisrelative amount could be a specific level of at least one bile acid.Alternatively, it could be a ratio of at least a first bile acid to atleast a second bile acid.

[0149] Non-limiting examples of suitable diseases are described ingreater detail below.

[0150] Colon Cancer

[0151] Coloscopic procedures are the accepted method for diagnosingColon cancer. Since this procedure is invasive and relatively costly,there is a need for diagnostic screening of colon cancer.

[0152] Research has shown a correlation in patients and controls betweenthe levels of unconjugated DCA in the serum and high colonicproliferation rates of colorectal mucosa—considered the first stages ofcancer (Ochsenkuhn T. et al., Cancer (1999), 85: 1664-1669). Additionalresearch has suggested that colon cancer risk can be identified bymonitoring bile acids and the changes in the ratio of DCA to cholic acid(Kamano T. et al. Dis Colon Rectum, (1999), 42(5):668-72).

[0153] GERD and Esophagus Cancer

[0154] One clinical approach being evaluated for patients with severeGERD and Barrett's esophagus is endoscopic surveillance to allow for thediagnosis of cancer at an early, treatable stage. For patients withoutdysplasia, endoscopic surveillance is recommended every 2-3 years andfor patients with dysplasia every 6 months. However, endoscopy has beenfound to miss cancer in a high proportion of cases. Currently, the onlymethod for detecting this condition is endoscopic surveillance.

[0155] Perhaps even more importantly, it is estimated that as many ashalf of the patients with Barrett's esophagus are asymptomatic. Thesepatients may either progress to cancer without treatment or are treatedat a late stage of cancer development. An alternative, more convenient,less intrusive and costly screening test is needed.

[0156] Monitoring the levels of secondary bile may prove effective as afirst level screening of patients with Barrett's esophagus to identifyhigh risk cancer patients or patients with GERD including asymptomaticpatients that otherwise may not be detected until cancer has developed.Possible methods for the monitoring of bile levels include but are notlimited to: screening secondary bile acid levels (Nehra D., et al., Gut,(1999); 44:598;Kauer W. K. H. et al., Surgery (1997); 122:874-81-whereconcentrations were found to be significantly higher than controls);detecting DCA and CDCA levels (Zhang et al., J Biol Chem (1998);273:2424-8; Shirvani et al. Gastroenterology 2000; 118: 487-496—whereDCA and CDCA were found to promote cancer development); the differencein ratios of conjugated to unconjugated bile acids (Shido K et al., Gut1998 42(2): 266-71; Thieson J. et al., J Gastointest Surg 2000 4(1):50-4—research showing proton pump inhibitor treatment leads to asignificant change in the ratio of conjugated to unconjugated bile).

[0157] It will be appreciated that the above descriptions are intendedonly to serve as examples, and that many other embodiments are possiblewithin the spirit and the scope of the present invention, as defined inthe claims which follow.

What is claimed is:
 1. A method for treating a subject for a diseaseselected from the group consisting of heart disease, gallstone disease,colorectal cancer, a precursor of colorectal cancer, gastroesophagealreflux diseases, esophageal cancer, COX-2 mediated inflammatoryconditions and cholestatic liver disease, the method comprising: orallyadministering a molecularly imprinted polymer to the subject.
 2. Themethod of claim 1, wherein the subject is treated for at least two ofthe above diseases concurrently.
 3. The method of claim 1, wherein saidmolecularly imprinted polymer is capable of binding to the toxin andsequestering the toxin.
 4. The method of claim 3, wherein saidmolecularly imprinted polymer comprises at least one functional monomerselected from the group consisting of: 2-, 3- and4-vinyl-2-hydroxypyridine and N,N′-diethyl(4-vinylphenyl)amidine.
 5. Themethod of claim 4, wherein said molecularly imprinted polymer furthercomprises a cross-linking agent for cross-linking said at least onefunctional monomer, wherein said crosslinking agent is selected from thegroup consisting of ethyleneglycol dimethacrylate, N,N′-diacryloyl- orN,N′-dimethacryloyl ethylenediamine, N,N′-diacryloyl- orN,N′-dimethacryloyl 1,3-diaminobenzene, N,N′-diacryloyl- orN,N′-dimethacryloyl 1,4-diaminobenzene, a diacrylate or dimethacrylateof 1,2-, 1,3-, or 1,4-dihydroxybenzene,N,N′-(4-vinylbenzoyl)-1,ω-diaminoalkane, and an N,N′-diacryloyl- orN,N′-dimethacryloyl 1,ω-diaminoalkane.
 6. The method of claim 3, whereinthe toxin comprises at least one bile acid or salt, or a combinationthereof.
 7. A method for treating a subject for a heart disease, themethod comprising: administering a molecularly imprinted polymer (MIP)compound to the subject.
 8. The method of claim 7, wherein the heartdisease is characterized by a condition selected from the groupconsisting of high cholesterol and oxidized LDL, or a combinationthereof.
 9. The method of claim 8, wherein said molecularly imprintedpolymer is capable of binding to the toxin and sequestering the toxin.10. The method of claim 9, wherein said molecularly imprinted polymercomprises at least one functional monomer selected from the groupconsisting of: 2-, 3- and 4-vinyl-2-hydroxypyridine andN,N′-diethyl(4-vinylphenyl)amidine.
 11. The method of claim 10, whereinsaid molecularly imprinted polymer further comprises a cross-linkingagent for cross-linking said at least one functional monomer, whereinsaid crosslinking agent is selected from the group consisting ofethyleneglycol dimethacrylate, N,N′-diacryloyl- or N,N′-dimethacryloylethylenediamine, N,N′-diacryloyl- or N,N′-dimethacryloyl1,3-diaminobenzene, N,N′-diacryloyl- or N,N′-dimethacryloyl1,4-diaminobenzene, a diacrylate or dimethacrylate of 1,2-, 1,3-, or1,4-dihydroxybenzene, N,N′-(4-vinylbenzoyl)-1,ω-diaminoalkane, and anN,N′-diacryloyl- or N,N′-dimethacryloyl 1,ω-diaminoalkane.
 12. Themethod of claim 9, wherein the toxin comprises at least one bile acid orsalt, or a combination thereof.
 13. A method for treating a subject fora disease of the gastrointestinal tract, the method comprising the stepof administering a molecularly imprinted polymer (MIP) compound to thesubject.
 14. The method of claim 13, wherein the disease of thegastrointestinal tract is selected from the group consisting ofcolorectal cancer, a precursor to colorectal cancer, gastroesophagealdisease, esophageal cancer, cholestatic liver disease and gallstonedisease.
 15. A method for treating a subject for a disease characterizedby a COX-2 mediated inflammatory condition, the method comprising thestep of administering a molecularly imprinted polymer (MIP) compound tothe subject.
 16. A method for performing combination therapy fortreating a subject for a disease, the method comprising the step ofadministering a combination of a molecularly imprinted polymer (MIP)compound and at least one additional drug to the subject.
 17. The methodof claim 16, wherein the disease is selected from the group consistingof heart disease, colorectal cancer, gastrointestinal reflux diseaseliver disease and a disease characterized by a Cox-2 mediatedinflammation.
 18. The method of claim 16, wherein said at least oneadditional drug alters at least one of the level or composition of thebile in at least a portion of the body.
 19. The method of claim 18,wherein said at least one additional drug is selected from the groupconsisting of a non-specific toxic bile acid or salt sequestrant,ursodeoxycholic acid and bile acid or salt transport inhibitors.
 20. Themethod of claim 16, wherein said molecularly imprinted polymer iscapable of binding to the toxin and sequestering the toxin.
 21. Themethod of claim 20, wherein said molecularly imprinted polymer comprisesat least one functional monomer selected from the group consisting of:2-, 3- and 4-vinyl-2-hydroxypyridine andN,N′-diethyl(4-vinylphenyl)amidine.
 22. The method of claim 21, whereinsaid molecularly imprinted polymer further comprises a cross-linkingagent for cross-linking said at least one functional monomer, whereinsaid crosslinking agent is selected from the group consisting ofethyleneglycol dimethacrylate, N,N′-diacryloyl- or N,N′-dimethacryloylethylenediamine, N,N′-diacryloyl- or N,N′-dimethacryloyl1,3-diaminobenzene, N,N′-diacryloyl- or N,N′-dimethacryloyl1,4-diaminobenzene, a diacrylate or dimethacrylate of 1,2-, 1,3-, or1,4-dihydroxybenzene, N,N′-(4-vinylbenzoyl)-1,ω-diaminoalkane, and anN,N′-diacryloyl- or N,N′-dimethacryloyl 1,ω-diaminoalkane.
 23. Themethod of claim 22, wherein the toxin comprises at least one bile acidor salt, or a combination thereof.